As our long-term goal, we will attempt to provide the understanding needed to design new polyketide synthases (PKSs) with novel properties, by characterizing the physical and functional nature of the fungal PKSs involved in the biosynthesis of the biologically active compounds mevinolin (lovastatin) and brefeldin A. We will strive to establish how these two multifunctional proteins, which are likely to contain sets of largely identical or very similar active sites and utilize the same two substrates, acetyl-and malonyl-Coenzyme A, are nonetheless able to catalyze the assembly of two distinct products with different carbon chain lengths, functionalization and cyclization patterns. Although we and others are studying different PKSs from bacteria as well as fungi for similar reasons, none of the mechanistic hypotheses emerging from these other studies are suited to the unique demands placed on the PKSs that are the focus of this proposal. The parallels between mevinolin and brefeldin A biosynthesis are expected to facilitate the understanding of the two PKSs by the construction of hybrid genes to make novel protein catalysts. In due course, we hope that the resulting knowledge will be instrumental in the design of novel protein catalysts that can make biotechnologically valuable compounds, including herbicides, insecticides, antiparasiticides, pharmaceuticals and biodegradable polymers, for many industrial uses. The research will be pursued in the following two laboratories. University of Wisconsin-Madison personnel will: 1) purify and physically characterize the mevinolin PKS, obtained by overexpression of the gene for this enzyme in a microbial host, and determine the structure of the product produced from acetyl-and malonyl-CoA in vivo and in vitro, through a collaboration with John Vederas at the University of Alberta; 2) extend the sequence analysis of the mevinolin gene cluster to include all of the genes for mevinolin biosynthesis; 3) clone and sequence the brefeldin A PKS gene(s) from P. brefeldianum DNA; and 4) initiate studies of the properties of mutuant and hybrid forms of the mevinolin PKS. University of Alberta, Canada, personnel will: 1) study the incorporation of various compounds into mevinolin or its precursor in vivo using a recombinant strain provided by UW-Madison; 2) if possible, extend this work in vitro using a cell-free system made from such a strain or the purified mevinolin PKS provided by UW-Madison; and 3) isolate and characterize any new intermediates of mevinolin biosynthesis uncovered.